Development of a simple HPLC-MS/MS method to simultaneously determine teriflunomide and its metabolite in human plasma and urine: Application to clinical pharmacokinetic study of teriflunomide sodium and leflunomide.

A simple high-performance liquid chromatography coupled with tandem mass spectrometry method was developed and fully validated to simultaneously determine teriflunomide (TER) and its metabolite 4-trifluoro-methylaniline oxanilic acid (4-TMOA) in human plasma and urine. Merely 50 μL plasma and 20 μL urine were employed in sample preparation using protein precipitation and direct dilution method, respectively. An Agilent Zorbax eclipse plus C column was selected to achieve rapid separation for TER and 4-TMOA within 3 min. Electrospray ionization under multiple reaction monitoring was used to monitor the ion transitions for TER (m/z 269.0 → 159.9), 4-TMOA (m/z 231.9 → 160.0), internal standard teriflunomide-d4 (m/z 273.0 → 164.0) and 2-amino-4-trifluoromethyl benzoic acid (m/z 203.8 → 120.1), operating in the negative ion mode. This method proved to have better accuracy and precision over concentration range of 10-5000 ng/mL in plasma as well as 10-10,000 ng/mL in urine. After a full validation, this method was successfully applied in a pharmacokinetic study of teriflunomide sodium and leflunomide in Chinese healthy volunteers.